Antibodies have shown great promise in the clinic for neutralizing a wide variety of target antigens. However, significant limitations have prevented monoclonal antibodies (mAbs) from realizing their full therapeutic potential. Antibody target neutralization and effector functions work best when multiple epitopes are targeted. Thus, mAbs have sub-optimal potency for many therapeutic applications. Oligoclonal combinations of mAbs targeting multiple epitopes have shown more promise, however, large doses are generally required, effector functions are compromised by the need for non-human hosts, and production costs are prohibitive for many applications. These limitations could be overcome by replacing conventional antibody effector function with proteolytic activities against target antigens. Proteolytic antibodies have been observed naturally, and certain disease states provoke the formation of antibodies with physiologically significant proteolytic activities, suggesting the potential efficacy of proteolytic antibodies in the clinic. The ability to harness the catalytic power of proteolytic antibodies for therapeutic uses could provide more potent pharmaceuticals than conventional antibodies that could be effective at lower doses. A third of the human germline kappa light chain variable domain (V() repertoire contain canonical serine protease-like catalytic triads in their complementarity- determining regions (CDR) and many have detectable peptidolytic activity. The tools of antibody engineering allow the construction of large libraries based on these human V( structures, which may contain effective proteolytic activities against a broad spectrum of therapeutic targets. What is lacking is a robust system for direct selection of desired activities from such libraries. To address this need, a proprietary cell-based system has been developed for selecting target-specific proteolytic activities with potentially unprecedented catalytic efficiencies from large libraries of proteolytic V( domains. The selection system comprises a proteolytic activity sensor in which an auto-inhibited reporter is linked to the target in such a way that cleavage of the target leads to activation of the reporter. When the sensor is expressed in E. coli cells along with a proteolytic V( library, cells expressing target-cleaving V( domains can be identified by the selectable phenotype conferred on the cells by the activated reporter. The system will be used to select and optimize human antibody light chains and Fab fragments containing V( domains with high proteolytic activity against Botulinum neurotoxin (BoNT). BoNTs are the most potent known toxins, and have been classified by the CDC as one of the six highest-risk agents for bioterrorism. Currently there are no effective small-molecule drugs for botulism, and due to the extreme lethality of BoNTs, it has not been possible to produce mAbs of adequate potency. In Phase I, human antibody Fab fragments that inactivate the catalytic subunit of BoNT will selected and optimized. In Phase II, the most active Fabs from Phase I will be subjected to pre-clinical evaluation in animals for both pre- and post- exposure safety and efficacy. [unreadable] PUBLIC HEALTH RELEVANCE: Monoclonal antibodies have shown great promise in the clinic, but significant limitations have prevented them from realizing their full therapeutic potential, including sub-optimal potencies and high costs. These limitations could be overcome by replacing conventional antibody effector function with proteolytic activities against target antigens. In this project, a system which has been developed for engineering proteolytic activity into antibodies will be applied to developing proteolytic antibodies against Botulinum neurotoxin, the most potent known toxin, and one of the greatest bioterrorism threats, for which there are no adequate treatments at present. [unreadable] [unreadable]